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, 284 (19), 13165-73

Selectivity of Docking Sites in MAPK Kinases

Affiliations

Selectivity of Docking Sites in MAPK Kinases

A Jane Bardwell et al. J Biol Chem.

Abstract

Protein kinases often recognize their substrates and regulators through docking interactions that occur outside of the active site; these interactions can help us to understand kinase networks, and to target kinases with drugs. During mitogen-activated protein kinase (MAPK) signaling, the ability of MAPK kinases (MKKs, or MEKs) to recognize their cognate MAPKs is facilitated by a short docking motif (the D-site) in the MKK N terminus, which binds to a complementary region on the MAPK. MAPKs then recognize many of their targets using the same strategy, because many MAPK substrates also contain D-sites. The extent to which docking contributes to the specificity of MAPK transactions is incompletely understood. Here we characterize the selectivity of the interaction between MKK-derived D-sites and MAPKs by measuring the ability of D-site peptides to inhibit MAPK-mediated phosphorylation of D-site-containing substrates. We find that all MKK D-sites bind better to their cognate MAPKs than they do to non-cognate MAPKs. For instance, the MKK3 D-site peptide, which is a remarkably potent inhibitor of p38alpha (IC(50) < 10 nm), does not inhibit JNK1 or JNK2. Likewise, MAPKs generally bind as well or better to cognate D-sites than to non-cognate D-sites. For instance, JNK1 and JNK2 do not appreciably bind to any D-sites other than their cognate D-sites from MKK4 and MKK7. In general, cognate, within-pathway interactions are preferred about an order of magnitude over non-cognate interactions. However, the selectivity of MAPKs and their cognate MKK-derived D-sites for each other is limited in some cases; in particular, ERK2 is not very selective. We conclude that MAPK-docking sites in MAPK kinases bind selectively to their cognate MAPKs.

Figures

FIGURE 1.
FIGURE 1.
MAPK pathways. The schematic shows six of the seven human MAPK kinases (MKKs, or MEKs) with their cognate, within-pathway MAPKs indicated by the arrows. The triangles on the MKKs represent their D-sites. See text for details.
FIGURE 2.
FIGURE 2.
Docking sites in MKKs. A, D-site peptides used in this study. Residues comprising the basic submotif (+++) are shown in bold and blue; residues comprising the hydrophobic-X-hydrophobic submotif (ϕXϕ) are shown in bold and red. Gaps have been introduced to maximize alignment of functionally similar residues; spaces are for visual clarity. The MEK1 peptide comprises residues 1-17 of the full-length protein; MEK2, 1-20; MKK3, 17-33; MKK6, 2-21; MKK4, 37-52; and MKK7-D2, 37-51. B, control peptides used in this study, with the wild-type version shown for comparison. Mutated residues are colored green.
FIGURE 3.
FIGURE 3.
D-site peptides from MKK3, MKK4, or MKK6 inhibit p38 phosphorylation of the MEF2A and ATF2 transcription factors. A, D-site peptides (triangle) were used to inhibit p38α phosphorylation of MEF2A or ATF2. B, purified GST-MEF2A (1 μm) was incubated with purified active p38α (22.5 nm) and [γ-32P]ATP for 20 min in the absence or presence of the specific concentrations of the indicated peptides. In the graph, results are plotted as percent phosphorylation relative to that observed in the absence of any added peptide. Phosphate incorporation into MEF2A was analyzed by SDS-PAGE and quantified on a PhosphorImager. Data are the average of at least three experiments, with duplicate data points in each experiment. To the right is shown is an autoradiogram of a representative experiment. C, as in B, except that the substrate was purified GST-ATF2 (1 μm). D, comparison of MKK3 and MKK4 peptides at lower doses. Reaction conditions are as in B, above.
FIGURE 4.
FIGURE 4.
D-site peptides from MEK1 and MEK2 are weak inhibitors of p38. A, D-site peptides (triangle) were tested for their ability to inhibit p38α phosphorylation of MEF2A. B, graph representing an average of multiple experiments, experimental details as in Fig. 3. C, an autoradiogram of a representative experiment.
FIGURE 5.
FIGURE 5.
The MKK3 and MKK6 D-site peptides are poor inhibitors of JNK1/2. A, D-site peptides (triangle) were tested for their ability to inhibit JNK1 or JNK2-mediated phosphorylation of ATF2 or c-Jun. B, purified GST-ATF2 (1 μm) was incubated with purified active JNK1 (2.5 nm) and [γ-32P]ATP for 20 min in the absence or presence of the specific concentrations of the indicated peptides. Other details are as in Fig. 3. C, as in B, except that the substrate was purified GST-c-Jun (1 μm), and the kinase was JNK2 (5 nm).
FIGURE 6.
FIGURE 6.
The MKK3 and MKK6 D-site peptides are effective inhibitors of ERK2. A, D-site peptides (triangle) were tested for their ability to inhibit ERK2-mediated phosphorylation of Elk-1. B, purified GST-Elk-1 (1 μm) was incubated with purified active ERK2 (∼1 nm) and [γ-32P]ATP for 20 min in the absence or presence of the specific concentrations of the indicated peptides. Other details are as in Fig. 3. C, an autoradiogram of a representative experiment. Elk-1 is phosphorylated by ERK2 on multiple residues, resulting in a ladder of bands displaying retarded electrophoretic mobility (36). Peptide-dependent inhibition was revealed both by the progressive collapsing of this ladder and by reduced phosphate incorporation overall. The fastest migrating band, which was largely unaffected by peptide inhibition, was excluded from quantification.
FIGURE 7.
FIGURE 7.
IC50 values for the inhibition of the given kinase/substrate pair by the indicated D-site peptide. The IC50 is the concentration of peptide required to inhibit kinase activity by 50%; a lower IC50 indicates better binding. Cognate interactions are highlighted in yellow, while non-cognate and control interactions are not highlighted.
FIGURE 8.
FIGURE 8.
Preferences of D-sites for MAPKs and of MAPKs for D-sites. A, all MAPKs that each D-site had been tested against are listed in order of preference, with highest to lowest binding affinity (i.e. lowest to highest IC50) running from top to bottom. The one or more cognate MAPK(s) for each D-site are indicated with blue text and an asterisk. Non-cognate MAPKs are indicated with red text. See Tables 1 and 2 for selectivity calculations. B, all D-sites that each MAPK had been tested against are listed in order of preference, as in A. Cognate D-sites for each MAPK are indicated with blue text and an asterisk. Non-cognate D-sites are indicated with red text.

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